Toxicological Assessment of Higher Olefins in OECD TG 422 Repeated Dose and Reproductive /Developmental Toxicity Screening Tests in Han Wistar Rats

Abstract

Higher olefins (HO) are used primarily as intermediates in the production of other chemicals, such as polymers, fatty acids, plasticizer alcohols, surfactants, lubricants, amine oxides, and detergent alcohols. The potential toxicity of five HO (i.e., 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene) with carbon ranging from C8 to C18 was examined in a combined repeated dose and reproduction/developmental toxicity screening study (OECD TG 422). These five HO were administered to Han Wistar rats by gavage at 0 (controls), 100, 300, and 1000 mg/kg bw/day. As a group of substances, adaptive changes in the liver (liver weight increase without pathological evidence), as well as increased kidney weight in male rats, were observed in HO with carbon numbers from C8 to C10. The overall systemic no observed adverse effect level (NOAEL) for all HO was determined at 1000 mg/kg bw/day. In the reproductive/developmental toxicity assessment, offspring viability, size, and weights were reduced in litters from females treated with Nonene at 1000 mg/kg bw/day. The overall no observed effects level (NOEL) for reproductive toxicity was considered to be 300 mg/kg bw/day for Nonene and 1000 mg/kg bw/day for the other four HO, respectively. These data significantly enrich the database on the toxicity of linear and branched HO, allowing comparison with similar data published on a range of linear and branched HO. Comparisons between structural class and study outcome provide further supportive data in order to validate the read-across hypothesis as part of an overall holistic testing strategy.

Keywords

higher olefins repeated dose toxicity OECD test guideline No. 422 no observed adverse effect level alkenes

Introduction

Higher olefins (HO) are versatile hydrocarbons and used primarily as intermediates for a number of important industrial and consumer products, such as polymers, fatty acids, plasticizer alcohols, surfactants, lubricants, amine oxides, and detergent alcohols.¹ Generally, they are alkenes with six to more than 30 carbons and a single double bond between two of the carbons (sum formula of C_nH_2n). HO demonstrate low acute toxicity by the oral, inhalation, and dermal routes of exposure, with no potential in genotoxicity and carcinogenicity.² Although HO are not eye irritants or skin sensitizer, prolonged exposure of the skin may cause skin irritation.²

Depending on the manufacturing processes, there are 4 general types of hydrocarbons that may be present in HO (Figure 1):

1. linear alpha olefins (i.e., vinyl—straight chain with a single double bond in the alpha position),

2. linear internal olefins (i.e., cis/trans disubstituted—straight chain with a single double bond in an internal position),

3. branched alpha olefins (i.e., vinylidene—isomerized olefins with a single double bond in the alpha position), and

4. branched internal olefins (i.e., trisubstituted and tetrasubstituted—isomerized olefins with a single double bond in an internal position).

Figure 1.

Various olefins and branching units referred to in the following as (A) Cis/trans disubstituted (linear internal); (B) Trisubstituted (branched internal); (C) Tetrasubstituted (branched internal); (D) Vinyl (linear alpha); and (E) Vinylidene (branched alpha).

The different positions of the double bond influence the metabolism of HO, which, in turn, may lead to differences in toxicity. Experimental studies in vitro demonstrated that olefins are oxidized by hepatic microsomal enzymes to glycols (diols) via an epoxide intermediate.³ While hydrolysis of the oxirane ring by epoxides hydrolase is rapid for alpha-olefins, glycol formation for internal- and branched-chain olefins is less efficient due to steric hindrance in the region of the oxirane ring by alkyl groups. This was illustrated for a series of C8-olefins, where the rate of hydrolysis of the epoxide intermediate showed the following trend⁴: 1,2 epoxy-n-C8 > 4,5 epoxy-n-C8 > 2,3 epoxy-iso-C8. In addition, other in vitro investigations, using hepatic microsomal fractions from phenobarbital-induced rats, have demonstrated loss of cytochrome P-450 due to alkylation of the haem function, with monooxygenase inactivation occurring to a greater extent in linear and branched alpha olefins relative to that found with internal olefins (with or without branching) where no or minimal loss of cytochrome P-450 was detected.⁵ Altogether, these results suggest that alpha olefins might be biologically more reactive than internal and/or branched olefins. Furthermore, in our previous HO bioavailability study, we applied in silico simulation which showed the predicted reactivity of HO decreased with the following order: linear alpha (vinyl) > branched alpha (vinylidene) > linear internal (cis/trans disubstituted) > branched internal (trisubstituted) > branched internal (tetrasubstituted).⁶ However, these results were based in vitro or in silico; meanwhile, we recognize and do not exclude the possibility that internal and branched molecules may induce systemic effects through mechanisms independent of epoxide formation in vivo. On the other hand, we also assessed the bioavailability of a series of HO via rat everted gut testing and concluded that the absorption of intestine is carbon number dependent with poor or barely no absorption if the carbon number is larger than 14 carbon.⁶ Based on this data, our hypothesis is that HO with low carbon number and linear alpha configuration may generate more (adverse) effects than the rest of the HO.

All in all, this paper summarizes the results of five repeated-dose studies, combined with reproductive/development toxicity screening (according to the OECD TG 422 (1996) study design⁷) in rats by gavage with HO carbon ranging from C8 to C18 and representing different types of HO, represented by selected chemicals listed in Table 1. By conducting these OECD TG 422 studies, we aim to (1) test the hypothesis which based on our previous in vitro and in silico data showed that short chain and alpha olefins would cause more adverse effects; (2) identify the hazard associated with the selected HO; and (3) compare all the findings within current studies and compare with other available studies to demonstrate a trend if any exist. In addition, these results significantly enrich the toxicological database of HO with an alpha or internal double bond and with linear and/or branched carbon chains. Furthermore, the results of this report provide a more comprehensive toxicity profile and the basis for comparison with data that has been reported previously on other members of the HO category effectively establishing a baseline.

Table 1.

Higher Olefins Used in the OECD 422 Studies.

Chemical Name	CAS Number	Carbon Number	Vinyl %	Cis/trans Disub %	Vinylidene %	Trisub %	Tetrasub %
1- Octene	111-66-0	8	∼90	<5	<5	0	0
Nonene	97280-95-0	9	0	∼30	<5	∼65	<1
Decene	25339-53-1	10	<5	∼90	<1	<5	<5
Hexadecene	26952-14-7	16	<1	∼70	<5	∼30	0
1-Octadecene	112-88-9	18	∼60	∼5	∼35	0	0

The highest concentration of any one structural element is marked in bold.

Materials and Methods

Test Materials and Dosing

Five test materials 1-octene (CAS No. 111-66-0), Nonene (CAS No. 97280-95-0), Decene (CAS No. 25339-53-1), Hexadecene (CAS No. 26952-14-7), and 1-Octadecene (CAS No. 112-88-9) were either obtained from a commercial source or lab-synthesized. A full list of test materials and their details are provided in Table 1. The selection of the test materials is based on three factors: commercial availability, carbon number, and the composition of the substances.

The test materials were prepared at appropriate concentrations by dissolving in Arachis oil BP (Evans Ltd., Liverpool, UK). The stability and homogeneity of the test material formulations were determined and confirmed to be stable for at least 21 days when stored at approximately 4°C in the dark. Formulations were therefore prepared fortnightly and stored at approximately 4°C in the dark under nitrogen. The analyzed concentrations of test material in the formulations were within the acceptable range (100% ± 10%) for the purposes of the studies.

The dose levels were selected based on available toxicity data including the results of 14-day dose range-finding (DRF) studies. In the DRF studies, no adverse effect of treatment had been apparent at doses up to 1000 mg/kg bw/day, which is the highest dose that is recommended in studies based on OECD TG 422 guideline. A high dose of 1000 mg/kg bw/day was therefore selected for these studies with 300 and 100 mg/kg bw/day as intermediate and low dose, respectively.

Animals and Animal Husbandry

All experiments were performed in compliance with UK Good Laboratory Practice (GLP) standards (Schedule 1, Good Laboratory Practice Regulations (SI 1999 No. 3106 as amended by SI 2004 No. 994)), which, in turn, are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17) and are also in accordance with the requirements of Directives 2004/9/EC and 2004/10/EC and the United Kingdom’s Animals (Scientific Procedure) Act 1986.^8,9

Male and female Wistar Han strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. The animals were acclimatized for eight days during which time their health status was assessed. At the start of treatment, the males weighed 311 to 358 g, the females weighed 192 to 234 g, and were approximately 12 weeks old. The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour, and the low-intensity fluorescent lighting was controlled to give 12 hours continuous light and 12 hours darkness. The temperature and relative humidity controls were set to achieve target values of 22 ± 3°C and 50 ± 20%, respectively.

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

The studies presented were performed in compliance with OECD Test Guideline No. 422: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996) (see Appendix Figure A1) at Harlan Laboratories Ltd., Shardlow, Derby, Derbyshire, UK.⁷

Briefly, groups of twelve male and twelve female animals were treated daily at the corresponding dose levels by oral gavage throughout the studies as shown in Table 2. All animals were observed for signs of functional/behavioral toxicity. These functional performance tests were developed from the methods used by Irwin¹⁰ and Moser et al.¹¹ Functional performance tests were performed on motor activity and forelimb/hindlimb grip strength according to the method employed by Meyer et al¹² and Reiter and MacPhail.¹³ Food consumption, water consumption, and body weight were measured at regular intervals throughout the administration and recovery periods. Following evidence of mating or at the end of the intended mating period, the males were returned to their original cages and females were transferred to individual cages. On completion of the mating phase, five randomly selected males per dose group were evaluated for functional/sensory responses to various stimuli. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, offspring surface righting, and offspring clinical signs were also recorded during this period. At Day 4 post partum, five randomly selected females per dose group were evaluated for functional/sensory responses to various stimuli.

Table 2.

Treatment Groups and Dose Level Used in the OECD TG 422 Repeated Dose Reproductive and Developmental Toxicity Screening Test of Higher Olefins.

Treatment Group	Dose Level (mg/kg bw/day)	Treatment Volume (mL/kg)	Concentration	Animal Numbers
Treatment Group	Dose Level (mg/kg bw/day)	Treatment Volume (mL/kg)	Concentration	Male	Female
Control	0	4	0	12	12
Low	100	4	25	12	12
Intermediate	300	4	75	12	12
High	1000	4	250	12	12

Hematological and clinical chemistry investigations were performed on five males and five females randomly selected from each test and control group prior to termination. Animals were not fasted prior to sampling. The following parameters were measured: hemoglobin (Hb), erythrocyte count (RBC), hematocrit (Hct), erythrocyte indices (i.e., mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC)), total leucocyte count (WBC), differential leucocyte count (i.e., neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), and basophils (Bas)), platelet count (PLT), reticulocyte count (Retic), prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (.11 mol/L). The following parameters were measured on plasma: urea, calcium (Ca++), glucose inorganic phosphorus (P), total protein (Tot.Prot.), aspartate aminotransferase (ASAT), albumin alanine aminotransferase (ALAT), albumin/globulin (A/G) ratio (by calculation), alkaline phosphatase (AP), Sodium (Na+), creatinine (Creat.), potassium (K+), total cholesterol (Chol), chloride (Cl-), total bilirubin (Bili), and bile acids.

Histopathological examinations were conducted on the preserved organs and tissues of five males and five females in the control and 1000 mg/kg bw/day dose groups. Samples of the following tissues were prepared: adrenals, ovaries, aorta (thoracic), pancreas, bone & bone marrow (femur including stifle joint), pituitary, bone & bone marrow (sternum), prostate, brain (including cerebrum, cerebellum, and pons), oesophagus, caecum, rectum, coagulating gland, salivary glands (submaxillary), colon, sciatic nerve, duodenum, seminal vesicles, epididymides (preserved in Modified Davidson’s fluid), Skin (hind limb), eyes (fixed in Davidson’s fluid), spinal cord (cervical, mid-thoracic and Gross lesions lumbar), heart, spleen, ileum (including Peyer’s patches), stomach, jejunum, thyroid/parathyroid, kidneys, trachea, liver, testes (preserved in Modified Davidson’s fluid), lungs (with bronchi) (were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), thymus, lymph nodes (mandibular and mesenteric), urinary bladder, mammary gland, uterus/cervix, muscle (skeletal), and vagina. In addition, sections of testes from all control and 1000 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined. Kidney sections from three males from each treatment group and control group were stained immunohistochemically for α-2u globulin (Rat α-2u globulin (AUG) Affinity Purified Polyclonal Ab, Batch No. AF586, R&D Systems Europe Ltd., 19 Barton Lane, Abingdon Science Park, Abingdon, OX14 3NB, UK) and examined.

Reproductive and Developmental Toxicity

The following reproduction and developmental parameters were recorded for each female and litter: date of pairing, date of mating, date and time of observed start of parturition, date and time of observed completion of parturition, number of offspring born, number of offspring alive, sex of offspring, clinical condition of offspring, and individual offspring weights. In addition, all live offspring were assessed for the physical development via surface righting reflex.

Statistical Analysis

The GraphPad prism software version 8.0 was used for graphical representation. Where appropriate, the homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variance was assessed using suitable ANOVA. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose-response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine the significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann–Whitney U test (non-parametric). Due to the preponderance of non-normally distributed data, reproductive parameters (implantation losses, offspring sex ratio, and offspring surface righting) were analyzed using nonparametric analyses. To detect the significance of intergroup differences from control, statistical significance was achieved at a level of P < .05.

Results and Discussion

For practical reasons and to keep the size of this manuscript manageable, we report herein only those effects and observations which are considered relevant to possible intrinsic hazardous properties of the test material. Effects such as spontaneous deaths, changed parameters within historical control ranges, changes that do not follow a dose-response, artifacts related to dosing procedures, and unaffected parameters are omitted. We believe that omission of this data in the summarized article does not compromise the interpretation of the results. However, to allow for a better overview, the findings based on data are summarized in Table 3.

Table 3.

Summary of Endpoints Conduced in OECD 422 Studies for 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene.

Endpoints	1-Octene	Nonene	Decene	Hexadecene	1-Octadecene
Parental Toxicity
Mortality	No unscheduled deaths.	No unscheduled deaths.	No unscheduled deaths.	No unscheduled deaths.	No unscheduled deaths.
Clinical observations	Either sex treated with 1000 mg/kg bw/day and males treated with 300 mg/kg bw/day showed episodes of increased salivation throughout the treatment period	Either sex treated with 1000 and 300 mg/kg bw/day showed episodes of increased salivation throughout the treatment period	Increased post-dosing salivation was observed for both sexes at 1000 mg/kg bw/day and, to a lesser extent, 300 mg/kg bw/day	At 1000 mg/kg bw/day was associated with increased post-dosing salivation for both sexes during the study.	Either sex treated with 1000 and 300 mg/kg bw/day showed episodes of increased salivation throughout the treatment period
Behavioral assessment	No treatment-related changes	No treatment-related changes	No treatment-related changes	No treatment-related changes	No treatment-related changes
Functional performance tests	No toxicologically significant changes	No toxicologically significant changes	No toxicologically significant changes	No toxicologically significant changes	No toxicologically significant changes
Sensory reactivity assessments	No treatment-related changes	No treatment-related changes	No treatment-related changes	No treatment-related changes	No treatment-related changes
Body weight	No treatment-related effects	Males treated with 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 and 3 of treatment. Overall body weight gain was reduced for these males	No treatment-related effects	No treatment-related effects	No treatment-related effects
Food consumption and food conversion efficiency	Unaffected	Unaffected	Unaffected	Unaffected	Unaffected
Water consumption	Increased water consumption for both sexes at 1000 mg/kg bw/day	Increased water consumption for both sexes at 1000 mg/kg bw/day and a slight increase in males treated with 300 or 100 mg/kg bw/day	Unaffected	Increased water consumption in males at 1000 mg/kg bw/day	Unaffected
Hematology	No toxicologically significant changes	Males treated with 1000 mg/kg bw/day showed a reduction in hemoglobin	No toxicologically significant changes	No toxicologically significant changes	No toxicologically significant changes
Blood chemistry	No toxicologically significant changes	No toxicologically significant changes	Higher glucose levels for males at 300 and 1000 mg/kg bw/day and higher cholesterol and alanine aminotransferase levels for females at 1000 mg/kg bw/day	No toxicologically significant changes	No toxicologically significant changes
Necropsy	One female at 1000 mg/kg bw/day had a thickened stomach	Nine males at 1000 mg/kg bw/day had enlarged kidneys, and 5 of them also had a dark liver. One male at 300 mg/kg had enlarged kidneys	No treatment-related findings	No treatment-related findings	No treatment-related findings
Organ weights	No toxicologically significant changes	Increase in absolute and relative liver weight for either sex at 1000 and 300 mg/kg bw/day and females at 100 mg/kg bw/day. Males from all treatment groups showed an increase in absolute and relative thyroid weight. Females treated with 1000 mg/kg bw/day showed a reduction in absolute and relative thyroid weight. Males treated with 1000 mg/kg bw/day also showed an increase in kidney weight both absolute and relative	Higher absolute and relative (to body weight) liver weights for both sexes at 1000 mg/kg bw/day and higher kidney weights for males at 1000 mg/kg bw/day	No toxicologically significant changes	No toxicologically significant changes
Histopathology	Stomach: both sexes at 1000 mg/kg bw/day show epithelia hyperplasia in the forestomach	Liver: both sexes hepatocyte hypertrophy at 1000 and 300 mg/kg bw/day and males at 100 mg/kg bw/day.	Stomach: both sexes at 1000 mg/kg bw/day and one male at 300 mg/kg bw/day show epithelia hyperplasia in the forestomach	Lung: both sexes at 1000 mg/kg bw/day show peribronchiolar inflammation	Mesenteric lymph nodes: inflammatory cell infiltrate was present in the periglandular fat surrounding the mesenteric lymph nodes in both sexes at 1000 and 300 mg/kg bw/day.
		Kidneys: proximal tubular basophilia and hyaline droplets in males from all dose groups, Tubular degeneration/debris in males at 1000 and 300 mg/kg bw/day.			Spleen: extramedullary hematopoiesis was greater in both sexes at 1000 mg/kg bw/day and in females at 300 mg/kg bw/day.
		Stomach: both sexes at 1000 mg/kg bw/day and males at 300 mg/kg bw/day show epithelia hyperplasia in the forestomach.			Lung: both sexes at 1000 mg/kg bw/day show peribronchiolar, granulomatous inflammation
		Thyroid: both sexes at 1000 and 300 mg/kg bw/day, and males at 100 mg/kg bw/day show follicular cell hypertrophy and hyperplasia.
		Pituitary: Increased incidence and severity of hypertrophic/vacuolated cells in pars anterior in males treated with 1000 and 300 mg/kg bw/day.
Reproductive and developmental toxicity
Mating	Unaffected	Unaffected	Unaffected	Unaffected	Unaffected
Fertility	Unaffected	Unaffected	Unaffected	Unaffected	Unaffected
Gestation lengths	Unaffected	Unaffected	Unaffected	Unaffected	Unaffected
Offspring litter size, sex ratio, and viability	No treatment-related findings	Litter size on Day 4 post partum was reduced in litters from females at 1000 mg/kg bw/day. A total litter loss was observed for one female at 1000 mg/kg bw/day.	No treatment-related findings	No treatment-related findings	No treatment-related findings
Offspring growth and development	No treatment-related findings	Offspring viability and litter weights on Day 4 post partum from females at 1000 mg/kg bw/day were reduced. Offspring body weight gain at 1000 mg/kg bw/day was reduced between Days 1 and 4 post partum.	No treatment-related findings	No treatment-related findings	No treatment-related findings
Offspring necropsy	No treatment-related findings	No treatment-related findings	No treatment-related findings	No treatment-related findings	No treatment-related findings

Parental Toxicity

Generally, for all five substances (i.e., 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene), no deaths related to treatment were observed in any of the dose groups (i.e. 0, 100, 300, and 1000 mg/kg bw/day). In detailed clinical observations, all substances induced episodes of increased salivation throughout the treatment period at the high dose. But in both males and females, no such effect was observed at 100 mg/kg bw/day. Increased salivation is considered to reflect distaste of the dosing formulations rather than any adverse systemic effect of treatment. The salivation incidence observed in these studies accompanied by increased water consumption is frequently observed when a slightly unpalatable or irritant test material is administered via the oral gavage route.¹⁴ Besides, no other treatment-related effects on behavioral assessment, functional performance tests (i.e., motor activity and forelimb/hindlimb grip strength), or sensory reactivity were observed in either sex for any substance at any dose level (Table 3).

Body Weight and Body Weight Gain

No statistical significance has been achieved for any of the HO when body weight gains were compared to controls for the various dose levels tested. Except for Nonene, none of the HO showed treatment-related effects on body weight gain in any dose group for either sex throughout the study, including gestation and lactation phases for females. Males treated with Nonene at 1000 mg/kg bw/day showed a statistically significant reduction in body weight gain during Weeks 1 (62%) and 3 (82%) of treatment, and overall body weight and body weight gain were reduced 6.7% (not statistically significant) and 38% (not statistically significant), respectively, when compared to control for these males (Figure 2). However, this effect is considered a result of local irritation of the digestive tract of the test material rather than a true effect of systemic toxicity. This assertion is supported by histopathological findings discussed further on. No reduced body weight gain effects were detected in treated females or males with Nonene at 300 or 100 mg/kg bw/day. In addition, females treated with 300 mg/kg bw/day Nonene showed a statistically significant increase in cumulative body weight gain during the final week of gestation. An increase in body weight gain is considered not to represent an adverse effect of treatment; therefore, the intergroup difference was considered not to be of toxicological importance.

Figure 2.

Body weight of animals orally administrated with Nonene (A) males, (B) females Pre-mating days, (C) females Gestation days, and (D) females Lactation days. Body weight gain of (E) males and (F) females. * compared to control, P < .05.

Food and Water Consumption

For all test materials, food consumption and food conversion efficiency were unaffected by treatment throughout the study, which included gestation and lactation phases for females at 100, 300, or 1000 mg/kg bw/day (Table 3). However, for water consumption during the pre-pairing phase, only the Decene and 1-Octadecene treatment groups did not reveal any significant difference in overall water consumption. Exposed to 1-Octene caused an increase in overall water consumption during the pre-pairing phase in animals of either sex treated with 1000 mg/kg bw/day; Nonene exposure also caused an increase in water intake at 1000 mg/kg bw/day in both sexes (approximately 53% for male and 111% for females) and a slight increase (approximately 13%) in males treated with 300 or 100 mg/kg bw/day throughout the treatment period; Hexadecene caused an increase in male rats only at 1000 mg/kg bw/day throughout the pre-pairing and post pairing phases of the study (Figure 3). As mentioned in section 3.1, increased water consumption is frequently observed when a test material which is slightly unpalatable or irritant is orally dosed.¹⁴

Figure 3.

Pre-mating daily water consumption of rat (both sex) orally administrated with 1-Octene, Nonene, Decene, Hexadecene, or 1-Octadecene.

Hematological and Blood Chemistry

No significant effects were detected in the hematological parameters examined in either sex for any substance, with the exception of Nonene, at 100, 300, or 1000 mg/kg bw/day (Tables 3 and 4). Nonene treatment resulted in a reduction in hemoglobin in males only (treated with 1000 mg/kg bw/day). It is noteworthy that this type of change (i.e., hematological parameters in particular hemoglobin) has been found commonly in males rather than females after exposure to hydrocarbons of this carbon range.¹⁵ Although the underlying mechanisms are unknown, these effects may be interpreted as (1) biological variations, because the differences are typically within normal physiological ranges and not associated with histological changes in the bone marrow or spleen; and/or (2) changes that are secondary to other treatment-related effects such as renal changes in males.¹⁶ Small reductions in hematological parameters are not unique to studies of hydrocarbons; rather, they are common findings in repeated dose studies and are considered to lack toxicological significance.¹⁷

Table 4.

Hematological Parameters of Parental Rats Receiving Nonene by Gavage.

Parameters	Nonene (mg/kg bw/day)
	Males				Females
	0	100	300	1000	0	100	300	1000
Hb (g/dl)	16.92 ± .44	16.86 ± .74	16.08 ± .31	14.94 ± 1.56 *	14.20 ± 1.02	14.14 ± .78	13.60 ± .80	14.06 ± .96
RBC (10^12/l)	8.98 ± .23	9.05 ± .42	8.77 ± .25	8.51 ± .82	7.30 ± .40	7.33 ± .42	6.85 ± .56	7.12 ± .52
Hct (%)	47.52 ± 1.20	47.96 ± 2.32	47.12 ± 1.34	44.38 ± 4.85	42.10 ± 2.18	41.54 ± 2.11	40.02 ± 2.69	41.34 ± 2.59
MCH (pg)	18.86 ± .44	18.64 ± .11	18.36 ± .48	17.58 ± .63*#	19.42 ± 1.03	19.24 ± .57	19.92 ± .70	19.74 ± .38
MCV (fl)	52.98 ± 1.38	53.00 ± 1.02	53.80 ± .97	52.12 ± 1.89	57.66 ± 2.29	56.68 ± 1.82	58.48 ± 1.89	58.08 ± 1.01
MCHC (g/dl)	35.66 ± 1.49	35.18 ± .67	34.12 ± .53**#	33.72 ± .28**#	33.68 ± .77	34.00 ± .20	34.00 ± .33	34.04 ± .27
WBC (10^9/l)	7.24 ± 1.13	8.70 ± 1.11	7.92 ± .80	7.26 ± 2.78	5.74 ± 1.76	6.48 ± .82	7.52 ± .82	7.00 ± 1.13
Neut (10^9/l)	1.56 ± .67	1.60 ± .22	1.22 ± .30	1.69 ± 1.14	1.36 ± .42	2.50 ± .57*	2.15 ± .46*	1.87 ± .51*
Lymph (10^9/l)	5.62 ± .69	7.06 ± 1.05	6.64 ± .95	5.48 ± 1.76	4.38 ± 1.55	3.97 ± .54	5.37 ± .57	5.11 ± 1.09
Mono (10^9/l)	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00
Eos (10^9/l)	.06 ± .08	.04 ± .06	.06 ± .07	.11 ± .12	.00 ± .00	.01 ± .03	.00 ± .00	.02 ± .04
Bas (10^9/l)	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00	.00 ± .00
CT (Seconds)	9.92 ± .47	9.40 ± .23*	9.34 ± .26*	9.34 ± .36*	9.24 ± .59	9.28 ± .94	8.90 ± .82	8.68 ± .37
PLT (10^9/l)	499.8 ± 129.8	593.6 ± 57.6	567.4 ± 34.6	540.6 ± 276	772.4 ± 141.6	808.4 ± 78.8	752.8 ± 338.7	845.6 ± 113.4
APTT (Seconds)	17.56 ± 1.63	16.62 ± .80	16.04 ± .76	16.70 ± 1.71	15.22 ± 3.99	16.98 ± 2.70	13.84 ± 3.85	15.40 ± 1.59

Note: Abbreviations used: Hb, Hemoglobin; RBC, Erythrocyte count; Hct, Hematocrit; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; WBC, Total leukocyte count; Neut, neutrophils; Lymph, lymphocytes; Mono, monocytes; Eos, eosinophils; Bas, basophils; CT, Prothrombin time; PLT, Platelet count; APTT, Activated partial thromboplastin time.

Values are group means and SD (n = 5 rats/sex). Probability values (P) are presented as follows: P < .01 **P < .05 *. #: values were within the historical control range.

Blood chemistry parameters were affected only in Decene treated in both sexes at all dose groups (Table 5), but these were of doubtful toxicological significance. For instance, males at 300 and 1000 mg/kg bw/day attained statistical significance in higher glucose levels when compared with control. However, in the absence of any histopathological correlates, this finding was considered unlikely to be of any great toxicological significance and not to represent an adverse effect of treatment. Females treated with Decene at 300 and 1000 mg/kg bw/day showed a statistically significant increase in total protein and albumin levels compared with control. However, the majority of individual values were within the historical control range, and no statistically significant differences from control were apparent for albumin/globulin ratio. Also, in the absence of any histopathological correlates, these findings were considered unlikely to be of any toxicological significance and not to represent an adverse effect of treatment. Other findings, including an increase in cholesterol and alanine aminotransferase, attained statistical significance at a dose level of 1000 mg/kg bw/day in females. Although the majority of individual values exceeded the historical control range, no histopathological correlates were observed. Therefore, these intergroup differences were considered unlikely to be of any toxicological significance and not to represent an adverse effect of treatment.

Table 5.

Biochemical Parameters of Parental Rats Receiving Decene by Gavage.

Parameters	Decene (mg/kg bw/day)
	Males				Females
	0	100	300	1000	0	100	300	1000
Urea (mg/dl)	46.6 ± 5.4	46.0 ± 2.9	50.2 ± 8.1	45.4 ± 5.7	44.4 ± 2.4	48.6 ± 6.8	54.6 ± 3.0**#	57.4 ± 4.4**#
Glucose (mg/dl)	155.0 ± 27.0	164.8 ± 17.7	177.6 ± 14.0*	183.6 ± 12.9*	134.6 ± 26.6	122.0 ± 17.8	136.2 ± 10.9	145.6 ± 11.8
Tot. Prot. (g/dl)	7.00 ± 1.09	6.68 ± .59	6.69 ± .71	6.75 ± .50	6.17 ± .23	6.40 ± .20	6.61 ± .38*	6.76 ± .41*
Albumin (g/dl)	3.70 ± .44	3.58 ± .25	3.58 ± .34	3.56 ± .30	3.48 ± .08	3.68 ± .19	3.78 ± .22*	3.82 ± .26*
A/G Ratio	1.14 ± .10	1.17 ± .05	1.16 ± .09	1.13 ± .13	1.30 ± .08	1.34 ± .12	1.34 ± .07	1.29 ± .03
Na+ (mmol/l)	145.8 ± 1.9	146.6 ± 3.2	143.4 ± 1.1	144.0 ± 0.7	145.0 ± 1.2	146.0 ± 2.7	146.2 ± 1.8	144.4 ± 2.4
K+ (mmol/l)	4.45 ± .45	4.29 ± .17	4.19 ± .33	4.57 ± .50	4.12 ± .16	4.28 ± .38	4.24 ± .28	4.00 ± .39
Cl- (mmol/l)	101.0 ± 1.2	102.0 ± 3.1	101.2 ± 0.8	99.6 ± 1.3	103.6 ± 1.5	103.0 ± 1.9	105.0 ± 1.4	102.4 ± 1.9
Ca++ (mmol/l)	2.76 ± .16	2.71 ± .12	2.71 ± .09	2.76 ± .08	2.70 ± .09	2.77 ± .09	2.69 ± .09	2.72 ± .11
P (mmol/l)	2.04 ± .48	1.90 ± .41	1.94 ± .22	1.96 ± .22	1.10 ± .29	1.10 ± .26	.84 ± .33	1.20 ± .37
ASAT (IU/l)	192.2 ± 231.4	127.8 ± 87.1	130.0 ± 123.1	82.0 ± 12.2	92.0 ± 21.7	102.6 ± 35.1	91.6 ± 26.1	88.2 ± 22.9
ALAT (IU/l)	74.8 ± 23.0	58.8 ± 8.2	63.2 ± 9.0	64.0 ± 11.3	62.8 ± 11.8	71.8 ± 7.3	73.2 ± 17.1	94.2 ± 21.9**
AP (IU/l)	181.6 ± 42.0	177.2 ± 58.6	133.0 ± 12.9	126.2 ± 54.8	193.4 ± 18.3	204.6 ± 34.8	134.0 ± 32.6*#	123.6 ± 53.4*#
Creat (mg/dl)	.74 ± .04	.73 ± .03	.73 ± .06	.72 ± .03	.80 ± .07	.75 ± .06	.78 ± .04	.76 ± .06
Chol (mg/dl)	101.2 ± 19.9	112.4 ± 20.7	112.0 ± 10.9	109.8 ± 18.3	63.4 ± 9.7	81.4 ± 20.9	78.4 ± 10.5	107.0 ± 15.1**
Bili (mg/dl)	.19 ± .09	.23 ± .08	.16 ± .03	.13 ± .03	.09 ± .02	.08 ± .02	.07 ± .02	.07 ± .01
Bile Acid (μmol/l)	9.42 ± 4.43	12.84 ± 5.99	9.14 ± 1.93	15.86 ± 8.18	10.24 ± 8.89	26.66 ± 29.90	14.54 ± 15.84	14.06 ± 9.57

Note: Abbreviations used: Tot. Prot., Total protein; A/G Ratio, Albumin/Globulin (A/G) ratio (by calculation); Na+, Sodium; K+, Potassium; Cl-. Chloride; Ca++, Calcium; P, Inorganic phosphorus; ASAT, Aspartate aminotransferase; ALAT, Alanine aminotransferase; AP, Alkaline phosphatase; Creat, Creatinine; Chol, Total cholesterol; Bili, Total bilirubin.

Values are group means and SD (n = 5 rats/sex). Probability values (P) are presented as follows: P < .01 **P < .05 *. #: values were within the historical control range.

Necropsy, Organ Weights, and Histopathological Findings

It is well recognized that the changes in organ weights together with findings in the necropsy and histopathological examinations are sensitive indicators of the detection of potentially toxic chemicals.¹⁸ Indeed, for all substances, variations as well as common effects were identified during necropsy, organ weights, and histopathological examinations in various treatment groups (Table 3). Thickened stomach was identified in one female rat after treatment with 1000 mg/kg bw/day 1-Octene during the necropsy. Together with epithelial hyperplasia in the forestomach identified at microscopic examination (Appendix Table A1), the stomach changes are considered to be an effect of treatment. While these microscopic findings probably represent an adverse effect of treatment, they are considered to reflect local irritation rather than any adverse systemic toxicity of the test material. In addition, humans do not have a physiological counterpart to the rat forestomach where substances may be present for a prolonged period of time; therefore, the macroscopic stomach changes detected have limited relevance to human toxicity. There were also changes identified in kidneys for one male rat at dose level of 1000 mg/kg bw/day and in lungs for one female rat treated with 300 mg/kg bw/day; however, the intergroup differences were considered not to be of toxicological importance in the absence of any histology correlates. In addition, other findings for 1-Octene exposure were not reported here since the values were well within the normal background range or had no associated histological findings. However, it is worth mentioning those results here so that we can have a better comparison of the observed effects in 1-Octene among other HO which will be discussed later. For instance, males treated with 1000 mg/kg bw/day showed an increase in liver and kidney weight both absolute and relative, males from all treatment groups also showed an increase in absolute and relative adrenal weight, and females treated with 1000 mg/kg bw/day showed an increase in absolute and relative thyroid weight. In terms of risk assessment, the findings observed on 1-Octene exposure would suggest that a NOAEL can be established at 1000 mg/kg bw/day because these findings were not evidence of true systemic toxicity based on the absence of any histology correlates.

Nonene, similar to 1-Octene, caused epithelial hyperplasia in the forestomach in animals of either sex dosed at 1000 mg/kg bw/day and in males at 300 mg/kg bw/day (Appendix Table A2). Indeed, Nonene caused local irritation of the forestomach. Interestingly, this local irritation effect does not influence the overall food consumption and overall body weight gain or body weight. This is because a rat stomach consists of two sections: a forestomach and a glandular stomach.¹⁹ The forestomach is anatomically and functionally distinct from the glandular stomach and serves as an aglandular food reservoir which transfers food to the glandular stomach to meet energy demands.^20-22 Besides, macroscopic findings detected at necropsy were confined to enlarged, pale, and mottled kidneys and a dark liver in a number of males treated with 1000 mg/kg bw/day, and enlarged kidneys for one male at 300 mg/kg bw/day. Microscopic examination also revealed effects in the kidneys of males from all Nonene treatment groups. Tubular basophilia and hyaline droplets were present in males from all Nonene treated groups. These tubular findings were also accompanied by tubular degeneration/debris in males treated with 1000 and 300 mg/kg bw/day. Tubular degeneration/debris may be considered to represent an adverse effect of the test material; however, immunohistochemical staining demonstrated that these findings were correlated to the same condition as hyaline droplets which are linked to the accumulation of alpha 2u-globulin. Nephropathy linked to alpha 2u-globulin is commonly observed and unique to male rats following treatment with some hydrocarbons and is not predictive of any adverse effect in humans.^23-26 Histopathological examination of the liver revealed hepatocellular hypertrophy in animals of either sex treated with 1000 and 300 mg/kg bw/day Nonene and in males treated with 100 mg/kg bw/day Nonene. Organ weight data supported this finding with increased absolute and relative liver weights observed in these animals (Table 6). The increased liver weights were interpreted by the pathologist as a compensatory effect due to increased metabolic demands.²⁷ Indeed, this interpretation was consistent with the results of the histological investigation which showed no degenerative or inflammatory changes and was supported by the fact that levels of liver enzymes markers (i.e. ALAT, ASAT, and AP) were not increased (Table 4). Therefore, this condition was considered to be adaptive in nature.²⁸ Furthermore, microscopic examination of the thyroid revealed increased incidence and severity of follicular cell hypertrophy/hyperplasia in animals of either sex treated with 1000 and 300 mg/kg bw/day and in males at 100 mg/kg bw/day. Males treated with 1000 and 300 mg/kg bw/day also showed hypertrophic/vacuolated cells in the pituitary. The thyroid, liver, and pituitary changes are characteristics of a consequence of hepatocellular induction as a result of enhanced hepatic metabolism.²⁹ As a side effect of hepatic induction, an increased liver metabolism of thyroid hormones T3 and T4 can occur. This subsequently leads to an enhanced thyroid gland production of these hormones, resulting in a negative feedback stimulation of TSH production. The appearance of thyroid follicular cell hypertrophy and hypertrophic/vacuolated cells in the pituitary are themselves considered to be a result of this process. The thyroid and pituitary changes were considered to be adaptive in nature. Based on the findings mentioned above and organ weight data (Table 6), a NOEL was therefore not established in either sex; however, in terms of risk assessment, these findings observed above would suggest that a NOAEL can be established at 1000 mg/kg bw/day for both sexes because the findings were not evident of relevant systemic toxicity.

Table 6.

Absolute Organ Weights in Rats Receiving Nonene by Gavage.

Organs	Nonene (mg/kg bw/day)
	Males				Females
	0	100	300	1000	0	100	300	1000
Terminal body weight (g)	394.8 ± 32.7	393.9 ± 26.1	398.4 ± 26.1	368.5 ± 26.0	274.6 ± 16.6	273.4 ± 12.8	281.2 ± 11.1	276.1 ± 10.8
Adrenals (g)	.07 ± .01	.07 ± .01	.07 ± .01	.07 ± .01	.08 ± .02	.09 ± .01	.09 ± .03	.09 ± .01
Bran (including cerebrum, cerebellum, and pons) (g)	2.04 ± .11	2.08 ± .08	2.06 ± .07	2.00 ± .08	1.86 ± .09	1.86 ± .11	1.88 ± .05	1.84 ± .07
Epididymides (g)	1.52 ± .20	1.50 ± .16	1.52 ± .19	1.41 ± .12
Heart (g)	1.10 ± .21	1.13 ± .15	1.06 ± .10	1.01 ± .10	.94 ± .07	.90 ± .14	.90 ± .15	.86 ± .08
Kidneys (g)	2.29 ± .33	2.31 ± .14	2.38 ± .16	3.17 ± .44**	1.55 ± .14	1.66 ± .12	1.66 ± .21	1.97 ± .12**
Liver (g)	12.78 ± 1.52	13.27 ± .98	14.39 ± 1.33**	19.34 ± 1.23**	10.54 ± .51	11.79 ± .53*	12.79 ± 1.30**	16.12 ± 1.62**
Ovaries (g)					.12 ± .01	.12 ± .01	.12 ± .02	.13 ± .03
Pituitary (g)	.01 ± .00	.01 ± .00	.01 ± .00	.01 ± .00	.02 ± .00	.02 ± .00	.02 ± .00	.01 ± .00**
Prostate (g)	.56 ± .08	.60 ± .10	.57 ± .09	.53 ± .06
Seminal vesicles (g)	1.93 ± .45	2.12 ± .45	2.10 ± .35	2.07 ± .34
Spleen (g)	.77 ± .12	.70 ± .11	.67 ± .11	.63 ± .08	.60 ± .05	.61 ± .09	.70 ± .09	.63 ± .11
Testes (g)	3.79 ± .47	3.65 ± .59	3.57 ± .31	3.51 ± .29
Thymus (g)	.42 ± .10	.40 ± .06	.39 ± .13	.30 ± .04	.28 ± .07	.23 ± .02	.25 ± .05	.22 ± .07
Thyroid/parathyroid (g)	.02 ± .00	.02 ± .00**	.02 ± .00**	.02 ± .00**	.02 ± .00	.02 ± .00	.02 ± .00	.02 ± .00*
Uterus & cervix (g)					.62 ± .10	.63 ± .10	.66 ± .10	.64 ± .07

Note: Values are group means and SD (n = 5 rats/sex). Probability values (P) are presented as follows: P < .01 **P < .05 *.

Oral gavage administration of Decene at 1000 mg/kg bw/day for both sexes did cause minimal to moderate epithelial hyperplasia of the forestomach (Appendix Table A3). Similar to other HO, this observation is likely to represent local irritation of the test material rather than any systemic toxicity. Also, macroscopic necropsy findings did not reveal any effects of Decene treatment, but absolute and body weight relative liver weights for both sexes at 1000 mg/kg bw/day were increased in comparison to control (Table 7). This increase may indicate an adaptive response in liver metabolic demand, and because there was no evidence of accompanying microscopic changes, it does not represent an adverse effect of treatment. A similar increase in absolute and body weight relative kidney weights for males at 1000 mg/kg bw/day was also observed (Table 7). Although differences from the historical control data were observed, in the absence of any evidence of histopathological change, it did not indicate an adverse effect. Besides these effects, males at 1000 mg/kg bw/day also showed statistically significantly lower absolute and relative spleen weights compared with control. However, all absolute and relative values for these treated males were within the historical control range, while control values occasionally exceeded the historical range. The differences in spleen weights were therefore considered incidental and unrelated to treatment, and this view was supported by the absence of any significant histopathological change for this organ. As a consequence, the NOAEL for adult systemic toxicity is considered to be 1000 mg/kg bw/day.

Table 7.

Absolute Organ Weights in Rats Receiving Decene by Gavage.

Organs	Decene (mg/kg bw/day)
	Males				Females
	0	100	300	1000	0	100	300	1000
Terminal body weight (g)	406.3 ± 22.7	410.5 ± 30.4	422.7 ± 12.6	396.1 ± 22.8	262.6 ± 16.3	262.3 ± 15.2	261.3 ± 11.9	260.6 ± 28.3
Adrenals (g)	.08 ± .02	.07 ± .02	.08 ± .02	.07 ± .03	.10 ± .01	.09 ± .01	.09 ± .02	.09 ± .00
Brain (including cerebrum, cerebellum and Pons) (g)	2.06 ± .06	2.00 ± .13	2.07 ± .08	2.03 ± .08	1.87 ± .05	1.86 ± .04	1.84 ± .10	1.81 ± .09
Epididymides (g)	1.54 ± .15	1.43 ± .28	1.47 ± .11	1.53 ± .16
Heart (g)	1.07 ± .08	1.11 ± .12	1.15 ± .10	1.08 ± .17	.86 ± .17	.88 ± .12	.88 ± .22	.86 ± .08
Kidneys (g)	2.45 ± .28	2.15 ± .06	2.58 ± .17	2.77 ± .38**	1.52 ± .17	1.60 ± .15	1.55 ± .13	1.65 ± .19
Liver (g)	13.05 ± .75	12.02 ± 1.28	14.75 ± .67	16.06 ± 2.21**	10.63 ± .83	10.93 ± 1.22	10.61 ± .71	12.22 ± 1.36*
Ovaries (g)					.12 ± .02	.12 ± .02	.12 ± .02	.11 ± .03
Pituitary (g)	.01 ± .00	.01 ± .00	.01 ± .00	.01 ± .00	.02 ± .00	.02 ± .00	.02 ± .00	.02 ± .00
Prostate (g)	.65 ± .16	.60 ± .08	.64 ± .15	.57 ± .09
Seminal vesicles (g)	1.95 ± .41	2.08 ± .50	2.09 ± .42	2.09 ± .29
Spleen (g)	.81 ± .10	.68 ± .09	.75 ± .05	.67 ± .08*#	.65 ± .04	.61 ± .03	.56 ± .03	.59 ± .11
Testes (g)	3.79 ± .19	3.90 ± .38	3.83 ± .28	3.81 ± .19
Thymus (g)	.44 ± .06	.34 ± .04	.38 ± .09	.41 ± .09	.22 ± .05	.22 ± .04	.24 ± .03	.23 ± .08
Thyroid/Parathyroid (g)	.02 ± .01	.02 ± .01	.02 ± .01	.02 ± .00	.02 ± .00	.02 ± .01	.02 ± .00	.02 ± .00
Uterus & cervix (g)					.72 ± .12	.74 ± .22	.60 ± .10	.61 ± .09

Note: Values are group means and SD (n = 5 rats/sex). Probability values (P) are presented as follows: P < .01 **P < .05 *. #: values were within the historical control range.

Macroscopic necropsy findings, organ weights, or histopathological examinations did not reveal any Hexadecene treatment-related effects identified in the stomach, liver, or kidney, but minimal or moderate peribronchiolar inflammation was observed in the lungs for both sexes at 1000 mg/kg bw/day (Appendix Table A4). The most likely explanation for this finding is that it arose from accidental aspiration of small amounts of the test material during the gavage procedure, which also been shown in other hydrocarbon oral gavage studies,³⁰ and it is considered very unlikely to have arisen as a consequence of systemic toxicity. As such, this dosage (i.e., 1000 mg/kg bw/day) represents a NOEL for adult toxicity and certainly is a clear NOAEL.

Unlike Hexadecene, the mesenteric lymph nodes and spleen showed significant changes after oral gavage with 1-Octadecene (Appendix Table A5). A minimal or mild inflammatory cell infiltrate was present in the periglandular fat surrounding the mesenteric lymph nodes (MLNs) in animals of either sex treated with 1000 and 300 mg/kg bw/day. The infiltrate was mixed in character, principally composed of lymphocytes and neutrophils, and tended to have a perivascular or perilymphatic orientation. The incidence and severity of extramedullary hematopoiesis was increased in the spleen in animals of either sex treated with 1000 mg/kg bw/day and in females treated with 300 mg/kg bw/day. The inter-group differences in females were not clearly dosage-related, and taking into account the low number of animals examined and the absence of supportive intergroup differences in hematological values or bone marrow findings, these differences were considered more likely to have arisen as a result of individual variation. Similar effects (i.e., histiocytosis in MLNs and spleen changes) were also observed with other similar molecular weight hydrocarbon molecules (i.e., mineral oil saturated hydrocarbons) after oral exposure of rats, and the joint FAO/WHO Expert Committee on Food Additives (JECFA 2012) considered that these effects observed in MLNs do not represent an adverse effect but rather should be considered a nonspecific, adaptive change of low toxicological concern.³¹ In addition, there were no statistically significant differences in adult organ weights that, in the absence of any evidence of histopathological change, were considered to be of any toxicological significance. In conclusion, a NOAEL can be established at 1000 mg/kg bw/day for both sexes because the findings were of low toxicological concern.

Reproductive and Developmental Findings

Except for Nonene, all HO (i.e., 1-Octene, Decene, Hexadecene, and 1-Octadecene) exerted no effects on reproduction, including the survival, growth, and development of the offspring in rats; therefore, the NOEL for reproductive toxicity for these substances was established at 1000 mg/kg bw (Table 3). Nonene treatment did not affect the mating performance and fertility (Table 8). Offspring viability (6/10) was, however, reduced in litters from females treated with Nonene at 1000 mg/kg bw/day on Day 4 post partum (Appendix Table A6). Subsequently, reduced litter size and litter weights were evident in these litters on Day 4 post partum when compared to controls. In addition, there is one female treated with 1000 mg/kg bw/day who had a total litter loss between Days 2 and 4 post partum, and this female was not included into the offspring viability calculation. Still, the mean offspring body weight gains for litters which survived to Day 5 post partum were reduced between Days 1 and 4 post partum at 1000 mg/kg bw/day. Therefore, based on the observation mentioned above, for Nonene the NOEL for reproductive toxicity was set at 300 mg/kg bw/day. Furthermore, whether the observed reduction in offspring viability observed with Nonene at 1000 mg/kg bw/day is a true hazard or not, needs further investigation as the viability index was driven by the litter of a single dam (Appendix, Table A6).

Table 8.

Reproductive/Developmental Parameters in Rats Treated Orally With Nonene.

Dose (mg/kg bw/day)	0	100	300	1000
Number of animals (females)	12	12	12	12
Number of paired (females)	12	12	12	12
Number of mated (females)	12	12	12	11
Number of pregnant (females)	11	11	12	11
Number of males paired	12	12	12	12
Precoital interval (days)	4.8±3.3	5.4±3.6	5.2±3.6	6.6±5.3
Mating index (%)	100	100	100	92
Pregnancy index (%)	92	92	100	100
Gestation length (Days)	22.7 ± 0.7	23.1 ± 0.6	22.9 ± 0.5	22.9 ± .05
Females with live offspring	11	11	12	11
Parturition index (%)	100	100	100	100
Number of corpora lutea	13.9 ± 2.3	14.2 ± 1.5	14.4 ± 1.8	14.0 ± 1.9
Number of implantation sites	12.1 ± 3.2	13.1 ± 2.7	13.9 ± 1.8	12.5 ± 3.3
Total number of offspring born	11.2 ± 3.4	11.6 ± 3.0	13.2 ± 1.9	11.1 ± 3.3
Rearing young to Day 5 of age	11	11	12	10
Offspring Weight change (g) Days 1 – 4	3.18 ± .9 (M), 3.07 ± .82 (F)	3.07 ± .67 (M), 3.02 ± .61 (F)	2.60 ± .62 (M), 2.54 ± .48 (F)	2.30 ± .85 (M)*, 2.42 ± .78 (F)
Pre-implantation loss (%)	13.9 ± 13.7	7.9 ± 16.2	3.4 ± 4.9	12.2 ± 18.6
Post-implantation loss (%)	8.5 ± 9.2	12.6 ± 15.3	5.4 ± 6.0	12.1 ± 8.2
Live birth index (%)	100 ± 0.0	100 ± 0.0	100 ± 0.0	97.1 ± 7.2
Viability index (%)	98.9 ± 3.8	99.2 ± 2.5	99.4 ± 1.9	91.9 ± 9.5*
Sex Ratio (% males) at birth	38.8 ± 21.3	49.0 ± 17.0	50.8 ± 12.2	52.2 ± 22.2

Mating Index (%), (Number of animals mated/Number of animals paired) × 100; Pregnancy Index (%), (Number of pregnant females/Number of animals mated) × 100; Parturition Index (%), (Number of females delivering live offspring/Number of pregnant females) × 100; Pre-implantation Loss (%), [(Number of corpora lutea – Number of implantation sites)/(Number of corpora lutea)] × 100; Post-implantation Loss (%), [(Number of implantation sites – Total number of offspring born)/(Number of implantation sites)] × 100; Live Birth Index (%), (Number of offspring alive on Day 1)/(Number of implantation sites) × 100; Viability Index (%), (Number of offspring alive on Day 4)/(Number of offspring alive on Day 1) × 100; Sex Ratio (% Males) at Birth, (Number of male offspring at birth)/(Total number of offspring at birth) × 100.

Values are means and SD.

Probability values (P) are presented as follows: P < .01 **P < .05.

Trend of Current Data and Compare with Other Publicly Available HO Data

The overall results show that all five tested HO have comparable levels of low toxicity in rats. Specifically, in 28 days repeated dose toxicity studies, 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene show a NOAEL for systemic toxicity at 1000 mg/kg bw/day. Although 1-Octadecene showed changes in the mesenteric lymph nodes and spleen (with a NOEL at 100 mg/kg bw/day), the observed differences were not considered to be of any toxicological significance in the absence of supportive evidence, for instance, hematological values or bone marrow finding correlations. In addition, there were typical effects observed after administration of the test materials due to the chemical properties. For instance, increased post-dosing salivation and water consumption due to distaste of the dosing formulations was seen in several instances. Also, common effects, such as microscopic changes in the forestomach and increased liver and kidneys weight, were only observed after administration of low carbon number alkenes (i.e., C8, C9, and C10) in contrast to high carbon number alkenes (i.e., C16 and C18). Besides the well-known irritative properties of low molecular weight alkenes, most of observed effects suggest that low carbon number alkenes are more readily metabolized than high carbon number alkenes. One of the possible explanations for the observed phenomenon is the bioavailability of the test material. Our previous in vitro gut absorption study showed that the carbon number, rather than other aspects of olefin structure (alpha or internal olefin; presence of branching; odd or even number chain length), determine the bioavailability after ingestion, suggesting that above a certain carbon number (∼C14) uptake from the gut is likely to be very low.⁶ Another aspect linked to bioavailability is the viscosity of the HO. According to the database of ECHA, a trend of increasing viscosity with increasing carbon number is seen in these types of substances. As carbon number increases so does the molecular weight so that viscosity acts as an indirect measure of molecular weight. Consequently and according to Kramers’ theory, increased chemical viscosity is associated with a decrease in the maximum rate of catalysis by enzymes.³² This might explain why some effects were only observed in low carbon number alkenes simply because high carbon number alkenes were metabolized slower than low carbon number alkenes or not at all. However, from the molecular weight point of view, comparing Nonene and Hexadecane (both Nonene and Hexadecane have similar composition (i.e., mainly Disub and Trisub)), the dose level of Nonene on a molar base is close to twice that of Hexadecane. The observed effects of Nonene treatment at 1000 mg/kg bw, which are absent with Hexadecane treatment, may be simply due to the dose level which is, on a molar basis, almost twice as high for Nonene than for Hexadecane. Overall, based on the NOAELs, all five HO appear not to be different with regard to repeated dose toxicity, while based on NOEL, low carbon number HO showed some effects due to higher bioavailability and irritation. For the reproductive/developmental toxicity, four HO (i.e., 1-Octene, Decene, Hexadecene, and 1-Octadecene) showed a NOEL at 1000 mg/kg bw/day, and only Nonene demonstrated reproductive toxicity effects at 1000 mg/kg bw/day based on offspring viability. However, according to the composition information (Table 1), a similar effect was expected to be observed with Hexadecane, whereas it has a NOEL for the reproductive/developmental toxicity at 1000 mg/kg bw/day. Therefore, bioavailability is not the only factor contributing to the reproductive toxicity effects, and other factors, such as the trisubstituted (branched internal) composition, may play a role.

Besides the data on the five HO reported herein, there are six other HO which has publicly available reports on sub-acute toxicity (i.e. OECD TG 407/OECD TG 422) and reproduction/developmental toxicity screening level (i.e., OECD TG 421/OECD TG 422), including 1-Hexene, alkenes C6, 1-Tetradecene, Alkenes C16-C18, Octadecene, and Alkenes C20-C24 (Table 9).^2,33-39 With regard to systemic toxicity, orally administrated 1-Hexene caused irritation of the gastric mucosa along with reduced body weights at the two top dose levels (i.e., 1010 and 3365 mg/kg bw/day) and spleen weights reduced at the top dose (i.e., 3365 mg/kg bw/day) for both sexes. In addition, male-rat specific kidney effects (i.e., hyaline droplet formation) were also observed at middle and top dose levels, resulting in a NOEL of 101 mg/kg bw/day for males and a NOAEL of 1010 mg/kg bw/day for both males and females. Alkenes C6 increased liver and kidneys weight at dose levels of 500 and 1000 mg/kg bw/day for both sexes and increased hyaline droplet formation in kidneys in males at 1000 mg/kg bw/day. Therefore, the NOAEL was set at 1000 mg/kg bw/day for both sexes, where the NOEL was determined at 100 mg/kg bw/day for females. 1-Tetradecene also showed increased liver weights at dose levels of 500 and 1000 mg/kg bw/day in both sexes. Then, the NOEL for females was set at 100 mg/kw bw/day but not determined for males due to the increased hyaline droplet formation in kidneys at all dose levels, leading therefore to a NOAEL of 1000 mg/kg bw/day for both sexes when considering that the kidney effects are male rat specific findings with no relevance for human health assessment. However, Alkenes C16-C18, Octadecene, and Alkenes C20-C24, all three members of the HO category showed no treatment-related adverse systemic toxicity effects, and therefore, the NOAEL was determined as 1000 mg/kg bw/day.

Table 9.

Other Sub-acute And/Or Reproduction/Developmental Toxicity Studies Tested on Higher Olefins Which Not Reported in Current Report.

Tested Substance	CAS Number	Type of Study	Species (Strain)	Route of Exposure	Composition	Dose Level (mg/kg bw/day)	NOAEL/NOEL	References
Alkenes, C6	68526-52-3	OECD 422	Rat (Sprague-Dawley)	Oral gavage	60% internal, branched	0, 100, 500, and 1000	NOAEL = 1000 mg/kg bw/day (systemic, reproduction, and developmental)	^2,35
1-Hexene	592-41-6	OECD 407	Rat (Wistar)	Oral gavage	90%–100% alpha olefins	0, 10, 101, 1010, and 3365	NOAEL = 1010 mg/kg bw/day (systemic)	^2,38
1-Hexene	592-41-6	OECD 421	Rat (Sprague-Dawley)	Oral gavage	90%–100% alpha olefins	0, 10, 500, and 1000	NOEL >1000 mg/kg bw/day (reproductive and developmental)	^2,40
1-Tetradecene	1120-36-1	OECD 421	Rat (Sprague-Dawley)	Oral gavage	Not reported	0, 100, 500, and 1000	NOAEL = 1000 mg/kg bw/day (reproductive and developmental)	^2,37
Alkenes C16-C18	Not reported	OECD 407	Rat (Sprague-Dawley)	Oral gavage	internal linear and branched olefin, 26% branched, linear internal 72%)	0, 25, 150, and 1000	NOAEL = 1000 mg/kg bw/day (systemic)	^2,39
Octadecene	182636-02-8	OECD 421	Rat (Sprague-Dawley)	Oral gavage	internal linear and branched, 32.5% branched	0, 100, 500, and 1000	NOAEL = 1000 mg/kg bw/day (reproductive and developmental)	^2,36
Alkenes C20-C24	C20 = 182636-03-9 C22 = 182636-04-0 C24 = 182636-05-1	OECD 407	Rat (Sprague-Dawley)	Oral gavage	branched and linear even-numbered carbon only) (>70% branched	0, 30, 300, and 1000	NOEL = 1000 mg/kg bw/day (systemic)	³⁴

OECD 422: Combined repeated dose toxicity study with reproduction/developmental toxicity screening test.

OECD 421: Reproduction/Developmental Toxicity Screening Test.

OECD 407: Repeated Dose 28-Day Oral Toxicity Study in Rodents

By comparing the results reported in the current report with previously published data, the irritative effects, as well as the increased liver and kidney weight observed with C6 and C14, were also confirmed in the present report for C8, C9, and C10. However, these observed effects are considered either adaptive (i.e., liver effects) or unrelated to humans (i.e., male rat kidney effects because of alpha-2-urinary globulin deposition). In addition, HO with carbon numbers > C16 showed nearly no adverse effect that are related to the test material in the sub-acute toxicity studies. Furthermore, the observed effects were rather dependent on the carbon numbers than the double-bond position (i.e., type of the HO). Altogether, based on the data reported in the current study, the trend of HO (i.e., from C6 to C24) for systemic toxicity, which has NOAEL values of 1000 mg/kg bw/day, is consistent.

For reproductive and developmental toxicity-related endpoints, no toxicologically meaningful differences were noted in F0 and F1 for all HO reported in Table 9 (i.e., 1-hexene, alkenes C6 1-tetradecene, and octadecene). Therefore, the NOAEL for HO reported in Table 9 regarding the reproductive and developmental toxicity was determined at 1000 mg/kg bw/day. By pooling all available HO reproductive and developmental toxicity data together (from C6 to C24), only C9 (i.e., Nonene) showed effects on developmental toxicity based on litter effects, including reduced size, weight, and viability at 1000 mg/kg bw. In addition, no trend was observed for reproductive and developmental toxicity either based on carbon number or type of double-bond. In order to further explore the observed effects of Nonene related to fetal development, a prenatal developmental toxicity study according to the OECD TG 414 guidelines and an extended one-generation reproductive toxicity study (EOGRTS) according to the OECD TG 443 guidelines are suggested to be conducted.

Conclusions

Our initial hypothesis was that HO with low carbon number and linear alpha configuration may generate more adverse effects than the rest of the HO. Based on evidence from the combined oral repeated dose and reproductive/development toxicity screening studies performed according to OECD TG 422, in rats, except for nonane, all other four tested HO were not considered to cause adverse systemic, reproductive, and developmental toxicity at dose levels up to 1000 mg/kg bw/day as no toxicologically significant effects on males or females were observed for any toxicologically relevant endpoint, even for the low carbon number and linear alpha configurations. Nonene consisting of highly branched molecules showed a NOEL of 300 mg/kg bw/day for reproductive toxicity. All observed effects in current study were consistent with previously published data on these substances or similar substances.

Pooling all available information, the generic effects of HO are (a) increased liver weight (adaptive liver response) observed from C6 to C14; (b) increased kidneys weight observed in males (hyaline droplets formation or alpha-2-urinary globulin nephropathy) from C6 to C14; (c) gavage induced forestomach local irritation observed from C6 to C10; (d) decreased gut absorption with increasing carbon chain length; (e) decreased severity of systemic effect by increasing carbon chain; and (f) Nonene (C9) seemingly an outlier showed effects on both systemic (i.e., stomach changes, organ weight, and microscopic findings in stomach, liver, kidney, thyroid, and pituitary) and reproductive toxicity with a NOEL of 300 mg/kg bw/day, indicating that at equivalent molecular weight basis, highly branched HO may cause more severe effects, apparently independent of the double bond position.

Footnotes

Acknowledgments

The authors would like to thank all members of the HOPA REACH Consortium (Higher Olefins and Poly Alpha Olefins REACH Consortium) and its members for helpful discussions and input during development of the manuscript and for assistance in preparation of the manuscript.

Author Contributions

The submitting author affirms that all individuals listed as authors agree that they have met the criteria of authorship and agree to the conclusions of the study. In order to meet the requirements of authorship, each author must have contributed to at least one aspect of each of the four criteria, as listed below. Please note that for Criteria 1 and 2, authors only to meet one of the two items listed. These criteria are not to be used as a means to disqualify colleagues from authorship who otherwise meet authorship criteria by denying them the opportunity to meet criteria 2 or 3. Therefore, all individuals who meet the first criterion should have the opportunity to participate in the drafting, review, and final approval of the manuscript. Any individuals not meeting the criteria may be mentioned in the Acknowledgements section of the manuscript.

Per the criteria defined by the International Committee for Medical Journal Editors (ICJME), please note the contribution made by each author listed in the manuscript. Please select items from the drop down menu.

Declaration of Conflicting Interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors of this article are either employed by companies that manufacture petroleum products or consultants.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The HOPA REACH Consortium (Higher Olefins and Poly Alpha Olefins REACH Consortium, website: ) and its members provided financial support for Penman Consulting staff and consultant participation, and the employers of the other authors provided salary and travel support in the normal course of their work.

Correction (November 2023):

In Table 1, there was a typo in the CAS no. of Nonene. The correct CAS no. is CAS 97280-95-0 and it has been corrected in the article.

ORCID iD

Quan Shi

Appendix

Table A1.

Summaries of Key Histopathological Findings in Rats Administered 1-Octene.

Parameters	Grade	1-Octene (mg/kg bw/day)
Parameters	Grade	0	100	300	1000
Male
Forestomach
- Hyperplasia, epithelial	++	0	0	0	4
- Vesiculation	+	0	0	0	3
- Inflammation; submucosal	+	0	0	0	2
- Congestion	+	0	0	0	1
- Edema	++	0	0	0	1
Liver
- Inflammatory cell foci	+	1	-	-	3
- Vacuolation; centrilobular	+	0	-	-	2
Kidneys
- Tubular basophilia	+	0	-	-	1
Lung
- Alveolar macrophages	++	3	-	-	1
- Inflammation; alveolar	+	1	-	-	-
Female
Forestomach
- Hyperplasia, epithelial	+	0	0	0	3
- Vesiculation	+	0	0	0	2
- Inflammation; submucosal	+	0	0	0	2
- Edema	++	0	0	0	1
Liver
- Inflammatory cell foci	+	1	-	-	3
- Vacuolation fine periportal	+	0	-	-	1
- Pigment; macrophages	+	0	-	-	1
- Hematopoiesis	+	3	-	-	1
Kidneys
- Tubular basophilia	+	0	-	-	1
- Mineral at corticomedullary junction	+	0	-	-	1
Lung
- Alveolar macrophages	+	2	0	0	2
- Hemorrhage	+++	0	1	0	0
- Congestion	++	0	1	1	0

-, Not examined; +, Minimal; ++, Slight; +++, Moderate.

Table A2.

Summaries of Key Histopathological Findings in Rats Administered Nonene.

Parameters	Grade	Nonene (mg/kg bw/day)
Parameters	Grade	0	100	300	1000
Male
Forestomach
- Hyperplasia, epithelial	+	0	0	2	2
Liver
- Hypertrophy; hepatic	++	0	5	5	9
- Congestion	+	0	0	0	5
Thyroid
- Hypertrophy/hyperplasia; follicular cell	+	2	5	5	5
Pituitary gland
- Hypertrophic/Vacuolated cells in pars anterior	++	10	9	12	9
Kidneys
- Tubular basophilia	++	0	3	4	9
- Tubular degeneration/debris	++	0	0	4	7
- Hyaline droplets	++	0	5	5	9
Kidney immunohistochemistry
- Alpha-2-microglobulin	++	3	3	3	3
Female
Forestomach
- Hyperplasia, epithelial	+	0	0	0	3
Liver
- Hypertrophy; hepatic	+	0	0	3	6
- Congestion	+++	0	0	0	1
Thyroid
- Hypertrophy/hyperplasia; follicular cell	+	2	2	3	5
Pituitary gland
- Hypertrophic/vacuolated cells in pars anterior	+	0	0	3	0
Kidneys
- Tubular basophilia	+	0	0	0	1
- Tubular degeneration/debris		0	0	0	0
- Hyaline droplets		0	0	0	0
Kidney immunohistochemistry
- Alpha-2-microglobulin		-	-	-	-

-, Not examined; +, Minimal; ++, Slight; +++, Moderate.

Table A3.

Summaries of Key Histopathological Findings in Rats Administered Decene.

Parameters	Grade	Decene (mg/kg bw/day)
Parameters	Grade	0	100	300	1000
Male
Forestomach
- Hyperplasia, epithelial	+	0	0	2	2
- Edema	++	0	0	1	0
Liver
- Inflammatory cell foci	+	3	-	-	2
- Brown pigment in hepatocytes	+	1	-	-	0
Kidneys
- Tubular basophilia	++	0	-	-	2
- Cyst; cortex	+	0	-	-	1
Spleen
- Hematopoiesis	+	4	-	-	3
- Hemosiderin	+	5	-	-	5
Female
Forestomach
- Hyperplasia, epithelial	++	0	0	0	4
Liver
- Inflammatory cell foci	+	1	-	-	3
- Vacuolation fine periportal	+	1	-	-	0
- Hematopoiesis	+	1	-	-	0
- Congestion	+	1	-	-	0
Kidneys
- Hydronephrosis	++	0	-	0	1
- Cyst; cortex	++	0	-	1	0
- Mineral at corticomedullary junction	+	1	-	0	1
- Brown pigment in cortical tubules	+	1	-	0	0
Spleen
- Hematopoiesis	+++	6	-	1	6
- Hemosiderin	++	6	-	1	4
- Congestion	+	0	-	1	0

-, Not examined; +, Minimal; ++, Slight; +++, Moderate.

Table A4.

Summaries of Key Histopathological Findings in Rats Administered Hexadecene.

Parameters	Grade	Hexadecene (mg/kg bw/day)
Parameters	Grade	0	100	300	1000
Male
Liver
- Inflammatory cell foci	+	2	-	-	1
Kidneys
- Unilateral hydronephrosis	+	1	-	-	0
Lungs
- Mononuclear cell infiltration, perivascular	+	1	0	0	0
- Inflammation, peribronchiolar	++	0	0	0	2
Female
Liver
- Inflammatory cell foci	+	0	-	-	1
- Extramedullary hematopoiesis	+	1	-	-	1
- Brown pigment, hepatocellular, periportal	++	0	-	-	1
- Congestion	+	0	-	-	1
Kidneys
- Inflammation, pelvis	+	1	-	-	0
- Hyperplasia, urothelium, diffuse	+	1	-	-	0
- Mineralization, cortico-medullary	+	1	-	-	1
- Hyperplasia, urothelium, focal	+	0	-	-	1
Lungs
- Mononuclear cell infiltration, perivascular	+	0	1	0	0
- Inflammation, peribronchiolar	++	0	0	0	3
- Alveolar macrophages, focal/multifocal	+	1	1	0	0

-, Not examined; +, Minimal; ++, Slight; +++, Moderate.

Table A5.

Summaries of Key Histopathological Findings in Rats Administered 1-Octadecene.

Parameters	Grade	1-Octadecene (mg/kg bw/day)
Parameters	Grade	0	100	300	1000
Male
Lymph node, mesenteric
- Intrasinusal congestion/erythrophagocytosis		1	2	0	0
- Inflammatory cell infiltrate, periglandular fat	++	0	0	3	3
Spleen
- Extramedullary hematopoiesis	+	1	0	0	3
Lungs
- Number of animals examined		5	-	-	5
- Mononuclear cell infiltration, perivascular	++	1	-	-	2
- Inflammation, peribronchiolar, granulomatous	++	2	-	-	3
Female
Lymph node, mesenteric
- Intrasinusal congestion/erythrophagocytosis		1	0	0	0
- Inflammatory cell infiltrate, periglandular fat	++	0	0	3	4
Spleen
- Extramedullary hematopoiesis	+++	4	4	5	4
Lungs
- Mononuclear cell infiltration, perivascular	+	2	-	-	0
- Inflammation, peribronchiolar, granulomatous	++	2	-	-	1

-, Not examined; +, Minimal; ++, Slight; +++, Moderate.

Table A6.

Individual and Group Mean Litter Size, Litter Weights, and Viability Index at 1000 mg/kg bw/day Nonene.

Animal Number	Number of Corpora Lutea	Number of Implantation Sites	Total Number of Offspring Born	Number of Live Offspring		Litter Weight (g)		Viability Index (%)
Animal Number	Number of Corpora Lutea	Number of Implantation Sites	Total Number of Offspring Born	Day 1	Day 4	Day 1	Day 4	Viability Index (%)
1	16	14	13	13	0	70.1	0	0
2	14	14	14	14	13	78.5	93.7	93
3	12	12	10	10	10	75.3	100.7	100
4	17	14	11	10	9	57.3	78.5	90
5	10	4	3	3	3	19.4	23.6	100
6	15	15	14	14	12	76.6	88.3	86
7	14	13	10	10	10	67.4	100.5	100
8	15	15	14	14	13	74.1	82.9	93
9	13	9	8	8	7	54	71	88
10	—	—	—	—	—	—	—	—
11	13	13	12	12	12	73.6	98.6	100
12	15	14	13	10	7	48.1	52.1	70
Mean	14	12.5	11.1	10.7	9.6	63.1	79	91.9
SD	1.9	3.3	3.3	3.3	3.2	17.7	24.7	9.5
N	11	11	11	11	10	11	10	10

SD: standard deviation; N: number.

Figure A1.

Diagram of the experimental schedule, indicating the maximum study duration, based on a full 14-day mating period. Adopted from OECD Guideline for Testing of Chemicals No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test 22.03.96.

Figure A2.

Group mean food consumption on both sex for 1-Octene, Nonene, Decene, Hexadecene, and 1-Octadecene.

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